Sequencing

:For the sense of "sequencing" used in electronic music, see the music sequencer article.

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In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.

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Overview

In genetics terminology, DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Currently, almost all DNA sequencing is performed using the chain termination methodhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=870828&query_hl=11, developed by Frederick Sanger. This technique uses sequence-specific termination of an in vitro DNA synthesis reaction using modified nucleotide substrates.

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Why sequence DNA?

The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in 'pure' research into why and how organisms live, as well as in applied subjects. Because of the key nature of DNA to living things, knowledge of DNA sequence may come in useful in practically any biological research. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for both genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology is a burgeoning discipline, with the potential for many useful products and services.

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Sanger sequencing

In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.

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The original Sanger sequencing method

There are two sub-types of chain-termination sequencing. In the original method, the nucleotide order of a particular DNA template can be inferred by performing four parallel extension reactions using one of the four chain-terminating bases in each reaction. The DNA fragments are detected by labelling the primer with radioactive phosphorous prior to performing the sequencing reaction. The four reactions would then be run out in four adjacent lanes on a slab polyacrylamide gel.

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A development of this method used four different fluorescent dye-labelled primers. This has the advantage of avoiding the need for radioactivity; increasing safety and speed, and also that the four reactions can be combined and run in a single gel lane, if they can be distinguished. This approach is known as 'dye primer sequencing'.

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Dye terminator sequencing

An alternative to the labelling the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to varying incorporation of the larger, bulkier chain-terminators interacting with the previously transcribed sequence of the template. To some extent this drawback has been reduced with modifications of the chemicals and enzymes used. This is the method now used in the vast majority of all sequencing reactions as it is both simpler and cheaper. In addition, the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this less of a concern with frequently used 'universal' primers.

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Automation and sample preparation

Modern automated DNA sequencing instruments are able to sequence as many as 96 fluoresecently labelled samples in a batch (run) and perform as many as 24 runs a day. These perform only the size separation and peak reading; the actual sequencing reaction(s), cleanup and resuspension in a suitable buffer must be performed separately.

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To produce detectable labelled products from template DNA, it is current practice to perform 'cycle sequencing'. By performing repeated (about 26) rounds of primer annealing, product extension and disassociation of DNA strands, the reactants are used more efficiently. This helps to reduce the cost. The different stages of the cycle are produced by altering the temperature of the reaction using a 'thermal cycler'. This relies on the fact that complementary DNA will anneal at a lower temperatures and disassociate at higher temperatures. An important part of making this possible is the use of DNA polymerase from a thermophillic organism, which is not rapidly degraded at the temperatures involved.

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Maxam-Gilbert sequencing

At the same time that the chain termination DNA sequencing method was introduced (1977), Maxam and Gilbert developed a second method of DNA sequencing based on chemical modification of DNA followed by its subsequent cleavage http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=265521. This method has fallen out of favor due to its technical complexity, the need for use of hazardous chemicals, and difficulties with scale-up.

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Relationship to sequencing strategies

DNA sequencing can only be used to determine relatively small regions of a particular DNA template at a time (a maximum of around 1000 base pairs on a modern DNA sequencer such as the Applied Biosystems ABI3730)http://www.appliedbiosystems.com/catalog/myab/StoreCatalog/products/CategoryDetails.jsp?hierarchyID=102&category1st=a50&category2nd=a51&category3rd=111907. In addition it much easier to get high quality sequence data when the region to be sequenced is purified, as it is on a PCR product or when a small region (a few kb) is cloned on a vector. To enable the sequence of complete genomes to be determined (which may contain more than 3000 million base pairs) a number of 'sequencing strategies' have been devised. Two of the most commonly used strategies are chromosome walking and shotgun sequencing.

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Other DNA sequencing methods

Other sequencing techniques which are under development, and may offer benefits over the conventional methods, include:

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• Sequencing by Hybridization
• Pyrosequencing

A novel method with incredible speed and accuracy.

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• nanopore sequencing

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